1. Field of the Invention
This invention relates to improved methods for transposon tagging and plant gene isolation. The specific improvements comprise efficient means for preselecting plants that have undergone transposition. Through use of this preselection, it is possible to substantially reduce the number of plants which must be inspected to locate individuals having the transposon excised and inserted into a gene of interest. The result is a substantial savings in time, costs and efficiency associated with growing and screening large numbers of plants for tagging and isolation of a desired plant gene.
Plants contain many genes having potential use for crop improvement. A limiting factor is the means to isolate useful genes. Many genes of interest, for instance genes encoding disease resistance, are difficult or impossible to isolate using standard biochemical or genetic engineering techniques.
One approach to gene isolation in plants has involved the use of transposable elements. A transposable element (TE) is a type of DNA sequence which has the ability to move or "jump" to new locations in the genome. A TE can insert into existing genes and by so doing cause mutation by gene disruption. TEs are known in bacteria, Drosophila, yeast, nematodes, plants and mammals and are believed to be ubiquitous.
A major limitation to transposon tagging as a means to isolate genes in plants is the fact that transposition in plants is a low frequency event. Typically, on the order of 1% of seed progeny may show evidence of transposition. Moreover, the chances of a TE landing in a particular gene of interest (for purposes of transposon tagging) is low because of the large number of DNA sequences into which transposition could take place. The overall frequency of detection of mutation due to TE insertion in a gene of interest is typically less than one in 10,000 (as a proportion of progeny seed screened). This means that well in excess of 10,000, and perhaps in the order of 100,000, plants may need to be screened for a high probability of identifying such a mutation.
2. Information Disclosure
Plant transposons are known. Transposon systems have been reported in Antirhinum majus, petunia and soybean. Nevers, P., et al., Ann Bot. Res., 12: 103-203, (1986). Corn is one of the better studied systems with several families of transposable elements having been investigated. Three families have received the most attention: the Ac/Ds system (McClintock, B., Cold Spring Harbor Symp. Quant. Biol., 21:197-216, 1956); the Spm/En system (McClintock, B., Carnegie Inst. Wash. Yrbook, 53:254-260, 1954); and the Mu system (Robertson, D.S., Mutat. Res., 51:21-28, 1978).
There have been reports that TEs, including Ac, can function (i.e., transpose) in plant species other than the one from which they originated. Baker, et al., PNAS USA 83: 4844-4848 (1986) and Van Sluys et al., EMBO J., 6:3881-3889 (1987).
The corn transposons Ac/Ds and Spm have been used for gene isolation in corn. Federoff, N., et al., 30 PNAS USA, 81:3825-3829 (1984) and Paz-Ares, J., et al., EMBO J., 5, 829-833(1986). Transposons have also been used to isolate genes from Antirhinum majus. Martin et al., EMBO J 4:1625-1630 (1985).
An assay permitting the selection in tissue culture for Ac transposition events has been reported Baker, B., et al., EMBO J 6:1547-1554, (1987). This assay involved placing the Ac DNA in the 5' untranslated region of a chimeric kanamycin resistance gene in a way which meant that the drug resistance gene was inoperative until Ac excision took place. Thus, they could select for Ac excision by placing transformed calli on kanamycin.